1. Field of the Invention
The present invention relates to the field of antiviral agents, and more specifically to their use in conjunction with known anti-cachexia agents. More particularly, the present invention relates to the potentiation of the antiviral activity of an antiviral agent (i.e., interferon or tumor necrosis factor) with an anti-cachexia agent (hydrazine sulfate) both in vitro and in vivo.
The present invention also relates to therapeutic treatments for the clinical management of viral infections. Methods for enhancing the in vivo antiviral activity of such agents as tumor necrosis factor and interferon are also included. Methods for preparing a therapeutic agent for the treatment of viral infections are also described.
2. Description of the Related Art
The behavior of tumor viruses both in vitro and in vivo.sup.40 has been studied in efforts to define the biology and molecular biology of cell transformation. Several types of tumor viruses, defined as viruses which have the ability to induce tumors experimentally in laboratory animals or to transform cells that are maintained in culture,.sup.27 have been linked as the causative agents of different cancers (Adenovirus (Ad12 and Ad18)) and herpesvirus infections (HSV-1 and HSV-2).
Research in the field of tumor and virus biology has provided critical insights regarding substances which affect their pathology. Two substances which appear to play important roles in tumor and viral growth and replication are tumor necrosis factor (TNF) and interferon (IFN).
Information gathered regarding tumor necrosis factor (TNF) and interferon (IFN) suggest they individually possess antiviral activity, making them potential candidates in the treatment of viral infections and tumors. However, serious side affects attendant treatment with therapeutically valuable doses of TNF or IFN limit their clinical usefulness. Thus, the employ of these agents in combating cancers and viral tumors has met with, at best, marginal success.
Additionally, it has been observed that patients with cancerous conditions typically suffer unexplained weight loss that is not controlled by antiviral agent treatments. This unexplained weight loss, or cachexia, is also a condition attendant several lethal viral infections, the most notorious of these being the HIV-induced infections (i.e., AIDS, ARC). Abnormal carbohydrate metabolism, as evident from changes in glucose production and glucose tolerance, has been cited as perhaps the precipitating condition facilitating this cachexia in cancer patients..sup.41
Tumor necrosis factor (TNF) has been shown to be produced by monocyte/macrophages in response to endotoxin and other stimuli. TNF has now been shown to have a
plethora of activities, which include antiviral activity,.sup.1 potentiation of interferon's antiviral activity,.sup.2 enhancement of monocyte motility,.sup.3 and activation of monocyte/macrophage cytotoxic activities..sup.4
TNF has also been shown to be identical to a known mediator of tissue wasting in neoplastic states,.sup.5 cachectin. TNF, like cachectin, is also known to promote tissue wasting processes. This wasting process greatly accelerates the morbidity and mortality of affected individuals. The mechanisms by which TNF/cachectin exert their effects are not entirely known, but it has been suggested that they are mediated by the suppression of lipoprotein lipase activity..sup.6 Serum cachectin levels have been directly correlated with tumor burden in sarcoma bearing experimental animals, and inversely with food intake and body weight..sup.7 Elevated levels of cachectin/TNF have also been associated with HIV-infected persons with AIDS or ARC, but not in asymtomatic HIV-infected persons..sup.8
Unfortunately, treatments with high dosages of tumor necrosis factor alone have been associated with such side affects as hypotension, leukocytosis, fever, chills, neurotoxicity, nausea and vomiting..sup.29 Moreover, high doses of IFN alone to cancer patients have been reported to demonstrate no therapeutic value..sup.29 These side affects are very similar, but more rapid, in onset than those observed with other biological response modifiers such as endotoxin and the interleukins.
Thus, the side affects of TNF has limited its use as an antiviral agent, despite its known antiviral activities, in addition to its host-destroying effects. It is uncertain, but these studies suggest tumor necrosis factor (TNF) may have intrinsic antiviral activity, as well as having the ability to potentiate the antiviral activity of interferon.
The interferons comprises a family of 20 to 25 low-molecular-weight proteins which cause cells to become resistant to the growth of a wide variety of viruses. The interferons can be divided into three species: IFN-alpha, IFN-beta, and IFN-gamma. The interferons differ in the agent which induces them and in the cell type which produces them.
Human IFN-alpha is produced by lymphocytes and macrophages which have been induced with components of foreign cells, and perhaps cells made foreign by transformation, infection and certain chemicals. Genetic sequences for at least 17 different IFN-alpha subtypes are encoded in human DNA. At least 8 are naturally expressed. The genes for IFN-alpha are all located in one region of human chromosome 9. IFN-alpha molecules are believed to be comprised of about 165-166 amino acids.
Human IFN-beta is produced by fibroblast and epithelial cells which have been induced with foreign nucleic acids. Genetic sequences for at least two IFN-beta subtypes are encoded in the DNA of humans. Both are expressed naturally. The gene for one of the IFN-beta subtypes has been mapped. It is located in close proximity to the IFN-alpha gene on human chromosome 9. The mature IFN-beta molecules are composed of about 166 amino acids. The two beta interferon subtypes, B.sub.1 and B.sub.2, also differ greatly in their activity. For example, interferon B.sub.1 has been found to have antiviral activity, while interferon B.sub.2 (alias interleukin-6).sup.42 does not have such antiviral activity. It should be noted that in the mouse system, IFNs alpha and beta are often produced concurrently.
IFN-gamma is produced by T-lymphocytes which have been stimulated with foreign antigens to which they have been previously sensitized, or with mitogens which stimulate this induction. Only one genetic sequence from IFN-gamma has been identified in the DNA of man. It is located on human chromosome 12. IFN-gamma molecules are composed of 146 amino acids but processing may alter this number.
As noted, interferon B.sub.1 is known to have antiviral activity. However, none of the interferons, to the Applicants' knowledge, have been associated with tissue-wasting or cachexia. The interferons were first recognized for their extraordinarily potent antiviral properties, and it has now been established that they may profoundly affect other viral, cellular and bodily functions, including cell metabolism and growth, immunity and tumor growth. Recent studies also demonstrate that interferons potentiate the cytotoxic effects of certain anti-neoplastic drugs on human tumor cells both in vitro and in vivo. However, the mechanism of interferon's synergistic action remains unknown..sup.28 Additionally, there is no clear-cut evidence that TNF is effective in the clinical setting.
Unfortunately, the interferons have been found to cause some undesirable side affects in vivo, such as fever, bone marrow suppression, fatigue, hypertension, malaise, nausea, anorexia, myalgia tachycardia, and impaired liver functions..sup.9, 29 Additionally, alpha-interferon, but not the other two classes of interferons, binds to opiate receptors in vitro, and causes endorphin-like opioid effects in vivo, including analgesia, lack of spontaneous locomotion, and catalepsy..sup.32
These prior studies, along with others.sup.9 establish that interferon is a useful therapy for particular conditions, particularly in cancer therapy, but cannot be used alone as a complete treatment regimen. Interferons (IFNs) have been tested in combination with other antitumor drugs in an effort to enhance antiviral activity and to reduce side effects associated with cancer chemotherapy. However, such has met with highly inconsistent results..sup.33, 34
One agent, hydrazine sulfate, has been identified as a potential therapeutic drug to treat the unexplained weight loss observed in cancer patients..sup.37 In particular, hydrazine sulfate has been shown to alter the abnormal carbohydrate metabolism described in the literature in cancer patients. (Id.) The potential of HS as an anti-cachexia agent is recognized as a consequence of its ability to inhibit gluconeogenesis, and particularly, the enzyme phosphoenolpyruvate carboxykinase..sup.10
Recently, hydrazine sulfate has been shown to influence whole body protein turnover in cancer patients..sup.37 The reduction in host weight loss (i.e., tissue wasting) exhibited in cancer and other tumor-bearing patients treated with hydrazine sulfate is described as a result of its effect on gluconeogenesis in the animal..sup.35 Additionally, hydrazine sulfate has little direct antiviral activity at low doses or at high doses to the Applicants, knowledge. Pretreatment with hydrazine sulfate has also been shown to protect against lethality after endotoxin challenge..sup.38
The use of hydrazine sulfate as an anticancer agent, in combination with another agent to enhance or potentiate its anticachexia affects has been considered by others. For example, the use of vitamin K.sub.2 or K.sub.3 (menadione) has been shown to synergize and/or potentiate the antitumor effect of hydrazine sulfate, while at the same time minimizing host toxicity (i.e., weight loss) and its symptomatically attendant tumorous conditions..sup.11, 12
It has been reported that hydrazine sulfate (HS), a non-competitive inhibitor of phosphoenolpyruvate carboxykinase in gluconeogenesis, can inhibit the wasting process in cancer patients..sup.10, 13 It was hypothesized that increased levels of lactic acid derived from highly glycolytic neoplastic tissue, amino acids from peripherial protein breakdown, and glycerol from lipid mobilization, contributed to the development of a greater than normal gluconeogenic pathway. This in turn resulted in greater amounts of energy being lost from normal host tissue to contribute to the obligatory production of glucose; hence, the wasting process. However, the possibility exists that HS might also be playing a role by inhibiting TNF/cachectin by some as yet undefined mechanism.
Some studies suggest that hydrazine sulfate as a component of tobacco smoke may be important in inhibiting interferon induction..sup.14 However, this affect was exhibited at only one 24 hour post-treatment time, with no affect being exhibited at any of the other treatment times examined..sup.14 While hydrazine sulfate has been shown to induce tumor regression and to inhibit tumor growth at 24 hours pretreatment, it has not as yet been shown to directly or indirectly inhibit TNF cytolytic activity or to potentiate TNF antiviral activity.
A combination therapeutic agent aimed at reducing the cytolytic action of these and other antiviral agents has not yet been examined. Moreover, the role of interferon in the management of various diseases has not yet been fully established.
Some investigators have examined the affect of administering TNF with such substances as adriamycin, a chemotherapeutic agent known to have antitumorigenic effects..sup.15 These particular studies found a synergistic enhancement of the adriamycin cytotoxicity in vitro, and further described a protective effect against host cachexia in vivo in animals treated with this combination..sup.15 However, no affect was described in regard to antiviral activity with this combination. Thus, the relatively high doses of TNF used thus far to elicit antiviral activity in an animal continues to limit the clinical utility of interferon owing to the undesirable side effects attendant these doses.
A combination therapeutic agent remains to be formulated which would enhance the antiviral activity of an antiviral agent to a level that would allow a reduction in currently used doses, thus minimizing the many in vivo side effects which have limited its clinical application. Ideally, such a combination would synergistically enhance the antiviral activity of the antiviral agent, and provide a protective function to the infected animal against continued host wasting (i.e. loss of host cell function and from the destruction of normal host cell integrity). Such an combination would present a significant advance in the clinical management of patients with viral and tumorous conditions, and presents a possible solution to the treatment of HIV infections such as AIDS, AIDS-related conditions and other viral infections, while halting the exacerbating and symptomatic tissue wasting attendant many virally-induced conditions.